Fig. 2.

Characterisation of rescued viruses in confocal microscopy and multistep growth study. (a) Expression of PPRV N, and H proteins and/or GFP with C77 mAb binding activity in the PPRV recombinants and parental virus infected cells. VDS cells were infected with viruses at MOI of 0.01 and fixed 24 h post-infection using 4% paraformaldehyde. Cells were stained separately with primary antibodies of mouse anti PPRV H (C77) and mouse anti PPRV N (C11) followed by secondary antibody Alexa Fluor 568 goat anti-mouse (red). Cell nuclei were stained with DAPI (blue) and GFP autoflorescence was visualised (green). The expression pattern of PPRV N and H proteins were comparable between the recombinant and parental virus. Wildtype H protein in PPRV Nigeria 75/1, rPPRV + GFP and rPPRV were detected using the C77 mAb whilst H was not detected using this antibody in cells infected with rPPRV-C77. The autoflorescence of GFP was detected for the rPPRV + GFP virus. (b) Growth kinetics study of recombinant and parental PPRVs in cell culture. Multi-step growth curve was obtained by infecting VDS cells with virus at an MOI 0.01 and grew for different time point and the titre of virus determined (TCID₅₀).